Characterization of genome-wide simple sequence repeats and application in interspecific genetic map integration in kiwifruit

Simple sequence repeats (SSRs) have been widely used in the construction of linkage maps, quantitative trait loci (QTLs) mapping, and marker-assisted selection (MAS). The availability of the sequenced Actinidia chinensis (kiwifruit) genome allows for the inexpensive and efficient development of microsatellite markers. In this study, a total of 49,067 SSRs were identified and characterized in the genome sequences of kiwifruit. Dinucleotide repeats are the most abundant SSRs, with the AG/TC motif accounting for 44.2 % of all SSRs in the genome. Fifty-five newly derived SSRs, together with 46 previously available SSRs, were integrated into linkage maps of an interspecific kiwifruit population. In addition, eight sex-linked SSR markers (including one previously published SSR) were mapped in the sex-related region on the LG25, suggesting that recombination is partially suppressed to maintain dioecy in kiwifruit. The SSRs developed from this study are a valuable resource for kiwifruit genetics and will contribute to the use of MAS in early sex determination of dioecious plant breeding.     

Assessing wheat (Triticum aestivum L.) genetic diversity using quality traits, amplified fragment length polymorphisms, simple sequence repeats and proteome analysis

The genetic diversity among 10 Iranian bread wheat (Triticum aestivum) genotypes was analysed using 12 quality traits, 320 amplified fragment length polymorphisms (AFLP) polymorphic fragments, 491 simple sequence repeats (SSR) alleles and 294 proteome markers. The results revealed that the genotypes differed for quality traits, AFLP, SSR and proteome markers. The average genetic diversity based on quality traits (0.684 with a range of 0.266–0.997) was higher than AFLP (0.502 with a range of 0.328–0.717), SSR (0.503 with a range of 0.409–0.595) and proteome (0.464 with a range of 0.264–0.870) markers. Although there were apparent similarities between the groupings of particular genotypes, the overall correspondence between the distance matrices appeared to be rather low. In this study, the cluster analysis based on AFLP data showed the closest agreement with genotypes’ regions of origin or pedigree information. In addition to the genetic diversity assessment, specific proteins with known function were detected uniquely for the studied genotypes. Our results suggest that the classification based on quality traits and genotypic markers of these wheat genotypes will be useful for wheat breeders to plan crosses for positive traits.     

Use of simple sequence length polymorphisms for genetic characterization of rat inbred strains

Genetic monitoring is an essential component of colony management and for the rat has been accomplished primarily by using immunological and biochemical markers. Here, we report that simple sequence length polymorphisms (SSLPs) are a faster and more economical way of monitoring inbred strains of rats. We characterized 61 inbred strains of rats, using primer pairs for 37 SSLPs. Each of these loci appeared to be highly polymorphic, with the number of alleles per locus ranging between 3 and 14 and, as a result, all the 61 inbred strains tested in this study could be provided with a unique strain profile. These strain profiles are also used for estimating the degree of similarity between strains. This information may provide the rationale in selecting strains for genetic crosses or for other specific purposes.     

Use of intercross outbred mice and single nucleotide polymorphisms to map skin cancer modifier loci

Car-R and Car-S outbred mouse lines, phenotypically selected for resistance and susceptibility to skin carcinogenesis respectively, show significant linkage disequilibrium (LD) at genetic markers mapping on chromosomal regions where skin cancer modifier loci (Skts3, Skts1, and Psl1 on Chrs 5, 7, and 9 respectively) have been mapped in standard crosses. Analysis of these regions for genetic linkage with skin cancer phenotypes in 245 (Car-R × Car-S)F₂ intercross mice, by using single nucleotide polymorphisms (SNPs), revealed significant linkage at a possible allelic form of the Skts1 locus, whose mapping region was shortened to a <5.5-cM interval near the Tyr locus. The Car-derived Skts1 locus was linked with papilloma multiplicity and latency by a recessive inheritance of the susceptibility allele. Putative loci on Chr 5 (Skts3) and 9 (Psl1) showed no significant linkage. These results point to the important role of the Stks1 locus in mouse skin tumorigenesis in independent crosses. The shortened Skts1 mapping region should facilitate the identification of candidate genes. Received: 23 June 2000 / Accepted: 22 November 2000     

Homogeneous genotyping assays for single nucleotide polymorphisms with fluorescence resonance energy transfer detection

A homogeneous detection mechanism based on fluorescence resonance energy transfer (FRET) has been developed for two DNA diagnostic tests. In the template-directed dye-terminator incorporation (TDI) assay, a donor dye-labeled primer is extended by DNA polymerase using allele-specific, acceptor dye-labeled ddNTPs. In the dye-labeled oligonucleotide ligation (DOL) assay, a donor dye-labeled common probe is joined to an allele-specific, acceptor dye-labeled probe by DNA ligase. Once the donor and acceptor dyes become part of a new molecule, intramolecular FRET is observed over background intermolecular FRET. The rise in FRET, therefore, can be used as an index for allele-specific ddNTP incorporation or probe ligation. Real time monitoring of FRET greatly increases the sensitivity and reliability of these assays. Change in FRET can also be measured by end-point reading when appropriate controls are included in the experiment. FRET detection proves to be a robust method in homogeneous DNA diagnostic assays.     

dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics

PCR-based detection of single nucleotide polymorphisms is a powerful tool for the plant geneticist. Cleaved amplified polymorphic sequence analysis is the most widely used approach for the detection of single nucleotide polymorphisms. However, this technique is limited to mutations which create or disrupt a restriction enzyme recognition site. This paper presents a modification of this technique where mismatches in a PCR primer are used to create a polymorphism based on the target mutation. This technique is useful for following known mutations in segregating populations and genetic mapping of isolated DNAs used for positional based cloning of new genes. In addition, a computer program has been developed that facilitates the design of these PCR primers.     

A simple procedure for the analysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis

We developed a modified allele-specific PCR procedure for assaying single nucleotide polymorphisms (SNPs) and used the procedure (called SNAP for single-nucleotide amplified polymorphisms) to generate 62 Arabidopsis mapping markers. SNAP primers contain a single base pair mismatch within three nucleotides from the 3' end of one allele (the specific allele) and in addition have a 3' mismatch with the nonspecific allele. A computer program called SNAPER was used to facilitate the design of primers that generate at least a 1,000-fold difference in the quantity of the amplification products from the specific and nonspecific SNP alleles. Because SNAP markers can be readily assayed by electrophoresis on standard agarose gels and because a public database of over 25,000 SNPs is available between the Arabidopsis Columbia and Landsberg erecta ecotypes, the SNAP method greatly facilitates the map-based cloning of Arabidopsis genes defined by a mutant phenotype.     

Simple sequence repeats for the genetic analysis of apple

 The development of highly informative markers, such as simple sequence repeats, for tagging genes controlling agronomic characters is essential for apple breeding. Furthermore the use of these markers is fundamental both for variety identification and for the characterisation and management of genetic resources. We have developed 16 reliable simple sequence repeat (SSR) markers that amplify all alleles from a panel of 19 Malus x domestica (Borkh.) cultivars or breeding selections and from Malus floribunda 821. Those markers show a high level of genetic polymorphism, with on average 8.2 alleles per locus and an average heterozygosity of 0.78. Due to this high level of polymorphism, it was possible using two selected SSRs to distinguish all cultivars except Starking and Red Delicious. Ten of the markers we developed have been mapped on a RAPD linkage map, proving their Mendelian segregation as well as their random distribution in the apple genome. Finally, we discuss the importance of using co-dominant markers in outbreeding species. Received: 8 October 1997 / Accepted: 9 December 1997     

Incidence,complexity and diversity of simple sequence repeats across potexvirus genomes

An in-silico analysis of simple sequence repeats (SSRs) in genomes of 32 species of potexviruses was performed wherein a total of 691 SSRs and 33 cSSRs were observed. Though SSRs were present in all the studied genomes their incident frequency ranged from 11 to 30 per genome. Further, 10 potexvirus genomes possessed no cSSRs when extracted at a dMAX of 10 and wherein present, the highest frequency was 3. SSR and cSSR incidence, relative density and relative abundance were non-significantly correlated with genome size and GC content suggesting an ongoing evolutionary and adaptive phase of the virus species. SSRs present primarily ranged from mono- to tri-nucleotide repeat motifs with a greatly skewed distribution across the coding and non-coding regions. Present work is an effort for the undergoing compilation and analysis of incidence, distribution and variation of the viral repeat sequences to understand their evolutionary and functional relevance.     

Using blocks of linked single nucleotide polymorphisms as highly polymorphic genetic markers for parentage analysis

Single nucleotide polymorphisms (SNPs) are plentiful in most genomes and amenable to high throughput genotyping, but they are not yet popular for parentage or paternity analysis. The markers are bi-allelic, so individually they contain little information about parentage, and in nonmodel organisms the process of identifying large numbers of unlinked SNPs can be daunting. We explore the possibility of using blocks of between three and 26 linked SNPs as highly polymorphic molecular markers for reconstructing male genotypes in polyandrous organisms with moderate (five offspring) to large (25 offspring) clutches of offspring. Haplotypes are inferred for each block of linked SNPs using the programs Haplore and Phase 2.1. Each multi-SNP haplotype is then treated as a separate allele, producing a highly polymorphic, 'microsatellite-like' marker. A simulation study is performed using haplotype frequencies derived from empirical data sets from Drosophila melanogaster and Mus musculus populations. We find that the markers produced are competitive with microsatellite loci in terms of single parent exclusion probabilities, particularly when using six or more linked SNPs to form a haplotype. These markers contain only modest rates of missing data and genotyping or phasing errors and thus should be seriously considered as molecular markers for parentage analysis, particularly when the study is interested in the functional significance of polymorphisms across the genome.     

Genetic diversity of two native sheep breeds by genome-wide analysis of single nucleotide polymorphisms

Wallachian and Sumava sheep are autochthonous breeds that have undergone a significant bottleneck effect and subsequent restoration efforts. The first objective of this study was to evaluate the degree of genetic variability of both breeds and, therefore, the current management of the breeding. The second was to determine whether these two breeds still retain their genetic uniqueness in relation to each other and other breeds, despite regenerative interventions. Our data consisted of 48 individuals of Sumava and 37 individuals of Wallachian sheep. The comparison data contained 25 other breeds (primarily European) from the HapMap dataset generated by the International Sheep Genomics Consortium. When comparing all 27 breeds, the Czech breeds clustered with 15 other breeds and formed a single branch with them according to Nei's distances. At the same time, however, the clusters of both breeds were integral and easily distinguishable from the others when displayed with principal component analysis (PCA). Population substructure analysis did not show any common genetic ancestry of the Czech national breeds and breeds used for regeneration or, eventually, breeds whose ancestral population was used for regeneration. The average values of F_ST were higher in Wallachian sheep (F_ST = 0.14) than in Sumava sheep (F_ST = 0.08). The linkage disequilibrium (LD) extension per autosome was higher in Wallachian than in Sumava sheep. Consequently, the N_e estimates five generations ago were 68 for Sumava versus 34 for Wallachian sheep. Both native Czech breeds exhibit a wide range of inbreeding based on the excess of homozygosity (F_HOM) among individuals, from ?0.04 to 0.16 in Sumava and from ?0.13 to 0.12 in Wallachian. Average inbreeding based on runs of homozygosity was 0.21 in Sumava and 0.27 in Wallachian. Most detected runs of homozygosity (ROH) were less than 5 Mb long for both breeds. ROH segments longer than 15 Mb were absent in Wallachian sheep. Concerning putative selection signatures, a total of 471 candidate genes in Wallachian sheep within 11 hotspots and 653 genes within 13 hotspots in Sumava sheep were identified. Czech breeds appear to be well differentiated from each other and other European breeds. Their genetic diversity is low, especially in the case of the Wallachian breed. Sumava is not so threatened by low diversity but has a larger share of the non-native gene pool.     

中国黄瓜主栽品种SSR遗传多样性分析及指纹图谱构建

应用简单重复序列(SSR)标记对从国内主要黄瓜育种单位征集到的116份生产上主栽品种进行遗传多样性分析。结果表明35对引物共扩增出86个等位基因,每对引物平均扩增出2.46个等位基因,其中有效等位基因占70.99%,平均Shannon’s信息指数为0.639,平均PIC为0.382。116个品种的遗传相似系数(GS)分布在0.5029~0.9797之间。聚类分析结果表明在遗传距离0.25处可将供试材料分为2大类群。第1类共106个品种,可分为5个亚族,主要包括华北密刺型、华南型、日本少刺型这3大类型;第2类包含10个欧洲温室型品种。     

Use of single nucleotide polymorphisms in candidate genes associated with daughter pregnancy rate for prediction of genetic merit for reproduction in Holstein cows

We evaluated 69 SNPs in genes previously related to fertility and production traits for their relationship to daughter pregnancy rate (DPR), cow conception rate (CCR) and heifer conception rate (HCR) in a separate population of Holstein cows grouped according to their predicted transmitting ability (PTA) [≤?1 (n = 1287) and ≥1.5 (n = 1036)] for DPR. Genotyping was performed using Sequenom MassARRAY^®. There were a total of 39 SNPs associated with the three fertility traits. The SNPs that explained the greater proportion of the genetic variation for DPR were COQ9 (3.2%), EPAS1 (1.0%), CAST (1.0%), C7H19orf60 (1.0%) and MRPL48 (1.0%); for CCR were GOLGA4 (2.4%), COQ9 (1.8%), EPAS1 (1.1%) and MRPL48 (0.8%); and for HCR were HSD17B7 (1.0%), AP3B1 (0.8%), HSD17B12 (0.7%) and CACNA1D (0.6%). Inclusion of 39 SNPs previously associated with DPR in the genetic evaluation system increased the reliability of PTA for DPR by 0.20%. Many of the genes represented by SNPs associated with fertility are involved in steroidogenesis or are regulated by steroids. A large proportion of SNPs previously associated with genetic merit for fertility in Holstein bulls maintained their association in a separate population of cows. The inclusion of these genes in genetic evaluation can improve reliabilities of genomic estimates for fertility.     

Discovery of single nucleotide polymorphisms in Lycopersicon esculentum by computer aided analysis of expressed sequence tags

Single nucleotide polymorphisms (SNPs) are useful for characterizing allelic variation, for genome-wide mapping, and as a tool for marker-assisted selection. Discovery of SNPs through de novo sequencing is inefficient within cultivated tomato (Lycopersicon esculentum Mill.) because the polymorphism rate is more than ten-fold lower than the sequencing error rate. The availability of expressed sequence tag (EST) data has made it feasible to discover putative SNPs in silico prior to experimental verification. By exploiting redundancy among EST data available for different varieties among 148,373 tomato ESTs, we have identified candidate SNPs for use within cultivated germplasm pools. 1,245 contigs having three EST sequences of Rio Grande and three EST sequences of TA496 were used for SNP discovery. We detected 1 SNP for every 8,500 bases analyzed, with 101 candidate SNPs in 44 genes identified. Sixty-six SNPs could be recognized by restriction enzymes, and subsequent experimental verification using restriction digestion or CEL I digestion confirmed 83% of the putative polymorphisms tested. SNPs between TA496 and Rio Grande have a high probability (53%) of detecting polymorphisms between other L. esculentum varieties. Twenty-six SNPs in 18 unigenes were mapped to specific chromosomes. Two SNPs, LEOH23 and LEOH37, were shown to be linked to quantitative trait loci contributing to fruit color within elite breeding populations. These results suggest that the growing databases of DNA sequence will yield information that facilitates improvement within the germplasm pools that have contributed to productive modern varieties.     

Mapping and analysis of simple sequence repeats in the Arabidopsis thaliana genome

Simple sequence repeats (SSRs) are becoming standard DNA markers for plant genome analysis and are being used as markers in marker assisted breeding. And hence because of its great significance we have initiated this study to analyze complete genome of Arabidopsis thaliana for the prevalence of mono-, di-, tri-, tetra-, penta- and hexa- mer repeats in the coding and non-coding regions of the chromosome and to map their exact position on the sequence. We have developed a program that can search a repeat of any length, its exact position on the chromosome and also its frequency of occurrence in the genome. Analysis of the results reveal that maximum number of repeats were found in chromosome 1 followed by chromosome 2 and 4 whereas, chromosome 3 and 5 contain relatively less number of these repeats. Among the SSRs, hexamers and dimers were more predominant in the chromosomes. Overall data showed that Chromosome 5 has minimum number of repeats. The abundance or rarity of various simple repeats in different chromosomes is not explained by nucleotide composition of sequence or potential repeated motifs to form alternative DNA structures. This suggests that in addition to nucleotide composition of repeat motifs, characteristic DNA replication / repair / recombination machinery might play an important role in genesis of repeats. The positional information is given at www.geocities.com/amubioinfo/ARD. This positional information can help Arabidopsis researchers to identify new polymorphisms in chromosomal regions of interest based on the SSRs that map in the area.     

Mining for single nucleotide polymorphisms and insertions/deletions in maize expressed sequence tag data

We have developed a computer based method to identify candidate single nucleotide polymorphisms (SNPs) and small insertions/deletions from expressed sequence tag data. Using a redundancy-based approach, valid SNPs are distinguished from erroneous sequence by their representation multiple times in an alignment of sequence reads. A second measure of validity was also calculated based on the cosegregation of the SNP pattern between multiple SNP loci in an alignment. The utility of this method was demonstrated by applying it to 102,551 maize (Zea mays) expressed sequence tag sequences. A total of 14,832 candidate polymorphisms were identified with an SNP redundancy score of two or greater. Segregation of these SNPs with haplotype indicates that candidate SNPs with high redundancy and cosegregation confidence scores are likely to represent true SNPs. This was confirmed by validation of 264 candidate SNPs from 27 loci, with a range of redundancy and cosegregation scores, in four inbred maize lines. The SNP transition/transversion ratio and insertion/deletion size frequencies correspond to those observed by direct sequencing methods of SNP discovery and suggest that the majority of predicted SNPs and insertion/deletions identified using this approach represent true genetic variation in maize.     

Development of cost-effective single nucleotide polymorphism marker assays for genetic diversity analysis in <Emphasis Type="Italic">Brassica rapa</Emphasis>

Competitive allele-specific PCR (KASPar) assay is a user-friendly system that provides flexibility in the numbers of single nucleotide polymorphisms (SNPs) and genotypes. Based on Illumina-GA-IIx genomic data from 10 genotypes with a broad genetic background, 3183 SNPs were selected for KASPar assays development, and 568 were finally converted and selected for Brassica rapa germplasm characterization (17.8%) on the basis of reproducibility, missing data rate, and uniform genetic distribution. High levels of polymorphism of these markers across 231 B. rapa genotypes were verified, illustrating by high polymorphic information content (averaged 0.34), minor allele frequency (0.37), genetic diversity (0.45), and the low observed heterozygosity (0.10). Based on the SNP dataset, structure and principal coordinates analysis, and neighbor-joining phylogenetic methods were used to examine the population structure and gave highly consistent results. The 231 accessions were divided into the four primary subspecies, representing 99 accessions from B. rapa ssp. pekinensis, 85 from B. rapa ssp. chinensis, 30 from B. rapa ssp. rapifera, and 17 from B. rapa ssp. oleifera and were further subdivided into 12 lower-order clusters according to different morphotypes. The genetic variability and pairwise fixation index analysis revealed that the ssp. pekinensis accessions possess the most extensive genetic variation among the four subspecies. The KASPar system is highly useful for validating SNPs and will be valuable for genetics research and breeding applications in B. rapa.     

Analysis of the genetic diversity of garlic (Allium sativum L.) by simple sequence repeat and inter simple sequence repeat analysis and agro-morphological traits

The genetic diversity of 39 garlic accessions was investigated using eight simple sequence repeat (SSR) primer combinations and 17 inter-simple sequence repeat (ISSR) primer combinations. A total of 109 polymorphic loci were detected among these accessions, with an average of 4.63 polymorphic loci per SSR primer combination and 4.29 polymorphic loci per ISSR primer combination. The mean effective number of alleles, the mean Nei's genetic diversity, and the mean Shannon's information index for SSR were 1.4799, 0.2870, and 0.4378, respectively; and those for ISSR were 1.4847, 0.2898 and 0.4415, respectively. Cluster analysis, using the unweighted pair-group method with arithmetic averages (UPGMA) based on the allele frequency data, classified the accessions into three groups. The results of principal component analysis (PCA) were consistent with those of the cluster analysis. PCA showed that each of these three groups exhibited significant variation in agro-morphological traits. These findings suggest that the eight SSR and 17 ISSR primers identified could define valuable markers for genetic diversity for use by plant breeders.